Download Early Changes in the Dynamics of Crypt Cell

[CANCER RESEARCH 37, 1680-1685, June 1977]
Early Changes in the Dynamics of Crypt Cell Populations in
Mouse Colon following Administration of
1,2-Dimethylhydrazine1
Thomas C. Richards
Department of Anatomy, School of Medicine, University of Oregon Health Sciences Center, Portland, Oregon 97201
SUMMARY
The effects
of the
carcinogen,
1 ,2-dimethylhydrazine
(DMH), on the proliferative characteristics of the crypt cell
population of mouse colon were studied. DMH (20 mg/kg
body weight) was injected s.c., weekly, for 2, 8, 16, 20, on 26
weeks. At the end of each treatment period, a group of
animals
was injected
with
[3Hjthymidine
and
killed.
After
2
weeks of DMH treatment, the cnypts appeared normal histo
logically,
but the total number
of cells, the number
of Ia
bebed cells, and the percentage of labeled cells pen crypt
column
had increased.
The
relative
distribution
of labeled
cells in crypt columns was not changed. DMH treatment did
not affect the phases of the cell cycle of epithelial cells and
the transit time of these cells through the crypt. None of the
indices of crypt dynamics were altered further with the
appearance of focal atypias (aften 16 weeks of DMH). How
ever, the total number of cells per crypt increased and the
percentage of labeled cells decreased as adenocarcinomas
developed in adjacent areas of the mucosa (after 20 to 26
weeks of DMH). The exact robe of these early mucosal
changes in the eventual development of malignant tumor
has
not
been
established.
However,
it appears
that
DMH
cancinogenesis
may involve two steps: (a) an initial increase
in the
of mitotically
number
active
cells
leading
to an en
weeks of s.c. injections of DMH (5, 20) and as early as 24 hr
after in. instillation of a single large dose of DMH (2). On the
other hand Maskens (13) reported an increase in both the
total number of cells per crypt and the number of labeled
cells pen crypt after 15 weeks of treatment. Localized patho
logical lesions (focal atypias) were consistently identified
after 12 to 16 weeks of treatment (5, 13, 20); however, a few
scattered atypias were noted on the tips of the folds of distal
colon as early as 38 days after initiation of treatment (5, 20).
Crypt cell populations in the colon are maintained in a
dynamic steady state by a balance between the production
of new cells (rnitosis) and the loss of old cells (extrusion) (4).
Disruptions in crypt dynamics that lead to the development
of an abnormal population, the appearance of focal atypias,
and eventually neoplastic growth may result from altera
tions in (a) the proportion of crypt cells that are mitotically
active, (b) the location of mitotically active cells within the
crypt, (c) the length of the cell cycle, on (d) the rate of cell
bossby extrusion (11). The purpose of this experiment was
to study the above indices of cell kinetics after selected
periods of weekly s.c. injections of DMH to document the
types of pnemabignant changes that may occur in Crypt cell
dynamics and to determine whether or not these changes
are present prior to the appearance of discrete lesions in the
mucosa.
bangedcell population; and (b) an eventual transformation
of at least some of the crypt cells of the enlarged
popula
tion.
MATERIALS AND METHODS
Animals and Treatments
INTRODUCTION
Several recent reports (5, 6, 20, 21) have shown that a
high incidence of adenocancinoma in distal colon can be
induced by s.c. injection of DMH2 into mice or rats at weekly
intervals for 20 to 30 weeks. However, because of different
experimental approaches used by various investigators, a
clean understanding of the premabignant changes that oc
cur in cobonic mucosa and of the time of their appearance
during the induction of cobonic cancer has not emerged.
The earliest change noted in the crypt cell population was a
doubling of the LI. This was reported to occur after 12
I This
work
was
supported
by
a
grant
from
the
Oregon
Division
of
American Cancer Society.
2 The
abbreviations
used
are:
DMH,
1 ,2-dimethylhydrazine;
index; i.r., intrarectal; [3H]TdR, tritiated thymidine.
Received September 7, 1976; accepted February 23, 1977.
1680
LI,
labeling
the
Female Swiss-Webster mice, at least 60 days old and 20 g
in body weight, were housed 5 animals/cage and were
given free access to food and water. The animals were given
weekly injections of DMH-HCI (20 mg/kg body weight;
American Scientific, Portland, Oreg.). DMH was dissolved
in 0.001 M EDTA, adjusted to pH 6.5 with sodium bicanbon
ate, and injected s.c. Controls were given comparable vol
umes of 0.001 M EDTA.
Animals were divided into 5 groups with at least 30 expeni
mentals and 30 controls in each group. DMH treatment was
initiated at the same time for all groups and was carried out
for 2, 8, 16, 20, or 26 weeks. One week after cessation of
treatment, the animals were killed and their colons were
analyzed. Several animals (3 to 7) were used to determine
changes in DNA-synthetic activity in cells of the crypt; each
animal was given an i.p. injection of [3H]TdR, 0.5 @Ci/g
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Crypt Cell Dynamics
and Colon Cancer
body weight (specific activity, 20 Ci/mrnole; New England
Nuclear, Boston, Mass.), and killed by cervical dislocation 1
hr later. The remainder of the animals were used to deter
mine the length of the cell cycle; they were given injection
of [3HJTdRand killed at intervals of 1 hr from 2 to 12 hr later
or intervals of 2 hr from 12 to 36 hr later. One group was
killed at 48 hr.
Tissue Handling
A segment
of distal
colon
at the
level
of the
pelvic
rim was
excised and used for analysis because previous studies
demonstrated that the descending colon was the most fre
quent site of tumor formation (5, 7). Care was taken not to
distort the colon during removal. A small section was ex
cised from the middle of the segment, washed thoroughly in
ice-cold 0.9% NaCI solution, fixed in Camnoy'sfluid for 2 hn,
and postfixed in 10% neutral-buffered formalin for 24 hr.
The tissue samples were embedded in paraffin and 5-pm
sections were prepared. Selected slides were prepared for
histological analysis and stained with hematoxylin and
eosin.
For madioautognaphic processing, 2 slides from each co
Ion were treated with 1 N HCI (60°)for 1 hr to hydrolyze the
nucleic acids and to remove any free labeled nucleotide
(18),
immersed
in Schiff's
reagent,
and
rinsed
in sodium
bisulfite (9). The slides were then dipped in NTB3 emulsion
(Eastman Kodak Co., Rochester, N. Y.), dried for 1 hn,
exposed for 3 weeks, and developed in Dektol (Eastman)
(15).
Analyses of Crypt Columns
Crypt columns, defined in these experiments as columns
of epithelial cells along 1 side of a mucosab crypt, were
selected for cell kinetic analysis if they were: (a) not dis
tonted by the presence of abnormal cell lesions (focal atyp
ias) or tumors, (b) sectioned longitudinally from top to
bottom, and (c) in contact with the musculamis mucosae.
The sections selected for examination were spaced so that
each crypt column was encountered only once. Fifty crypt
columns were analyzed for each animal.
LI. Four indices of kinetics of crypt cells were measured.
The length of the crypt column was determined by counting
the cell positions starting from the base of the crypt and
continuing up to the junction with surface cells (Chart 1;
Ref. 12). A cell was considered labeled if 3 or more grains
were seen over the nucleus. The number of labeled nuclei in
each cnypt was recorded. The LI was determined by calcu
bating the ratio (3).
@
Labeledcells in a population
Total cells in a population
@
Student's t test was used for comparisons between means
of values from DMH-tmeated and control animals (19).
Absolute and Relative Distribution of Labeled Cells In
Crypt Columns. The length of each crypt was normalized to
100 units because of the variations in crypt length in ani
mals from different treatments. For a determination of what
portion of the crypt contained the mitotically active cells,
Control
2 week DMH
treatment
26 week DMH
treatment
Chart 1. Distribution of proliferating cells in crypt columns of colons
from control animals and animals treated with DMH for 2- and 26-week,
periods. Dark cells, cells that are labeled 1 hr after injection with [3H]TdR.
Changes in the distribution of mitotically active cells were determined by
counting the numbers of labeled cells in quarters and segments of the
crypts. Division of the crypts into quarters and segments was based on the
relative lengths of the crypt column.
crypts were divided into quarters, and each labeled cell was
assigned to a specific quarter. An analysis of variance was
used to test for differences in values within each quarter of
the crypt. The Newman-Keuls multiple range test was used
to determine differences in values between the quarters of
the crypt. Differences between the means from control and
DMH-treated animals for each quarter were determined by
an analysis of variance.
The crypt was further divided into 10 equal segments to
localize more accurately changes in the distribution of mi
totically active cells; the percentage of labeled cells in each
crypt segment was calculated to determine whether on not
there was an increase in the proportion of cells that were
dividing. These data were transformed for statistical pun
poses by the arc sine of the square mootof the percentage,
and differences in distribution of labeled cells between con
tnol and DMH-tneated animals were determined by covani
ance analysis (19).
Length of the Cell Cycle. The phases of the cell cycle of
crypt cells were determined from a curve that was gener
ated by plotting the fraction of labeled mitoses against the
time after injection with [3H]TdR (17). At least 50 mitoses
from each animal were counted.
The curves for the control
and DMH-tmeatedanimals were fitted to the points by hand,
and individual pains of points along the curves were corn
pared statistically by Student's t test.
Rate of Migration In Crypt Columns. The velocity at
which cells moved upward in the crypt was deduced from
the distribution of mitotic cells in the crypt. The number and
position of all mitotic figures were recorded . It was assumed
that the length of mitosis was 1 hr at all levels of the crypt.
The rate of migration of cells at any position in the crypt was
considered to be equal to the cumulative rate of prolifera
tion up to that position. The mateof proliferation was deter
mined by the mitotic index of each position (1).
Proportion
of cells in a population
Mitotic time
that are mitotic
RESULTS
Gross and Microscopic Findings. The most striking result
observed in colons from animals treated with DMH for 2
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1681
T. C. Richards
weeks was an increased cellularity (compare Figs. 1 and 2).
Although only the changes in the crypts were quantitated,
there appeared to be an increase also in the number of cells
in the larnina pmopnia and submucosa. Crypt cell length
initially increased by about 25% over control values (Table
1). Subsequent treatments caused no further change in the
length of the crypts until after 26 weeks when the crypt
length increased by 40% oven controls. This marked in
crease in the total number of cells coincided with the ap
pearance of frank tumor in adjacent areas of the mucosa.
Focal atypias were first observed in scattered crypts after
16 weeks of DMH treatment (Fig. 3). The atypias, defined as
localized lesions of atypical cell structure and high mitotic
activity (5), were usually located in the outer or lurninab
halves of the cnypts. The lesions were irregular in shape and
occupied areas of the larnina propnia; they were not ob
served deep to the musculanis mucosae.
A few
small
but
grossly
visible
tumors
were
observed
in
the colons from some animals after 20 weeks of DMH treat
ment. Multiple barge lesions were observed throughout the
distal colon of almost all animals treated for 26 weeks with
DMH (Fig. 4). In many of the tumors examined histobogi
cabby,cells in the base of the lesion had invaded the submu
cosa; this was interpreted as evidence of cancer. Lange
ovemgrowths of cells blocked the lumens of many colons
examined.
Lb. The mean number of labeled cells per crypt column
and the LI increased after 2 weeks of DMH treatment
1). The
number
of labeled
cells
pen crypt
column
(Table
approxi
mately doubled compared to control values, whereas the LI
increased by a lessen amount. Neither of these values
changed further with subsequent treatment until frank tu
Table 1
Effects of DMH treatment on cells of crypt columns from distal
colon
Total number of cells (crypt length), number of labeled cells,
number of mitotic cells, and percentage of labeled cells per column
of crypt cells in histologically normal cryptsfrom control and DMH
treated
[3H]TdR.Length
animals killed 1 hr after i.p. injection of
of treat
ment
@
Crypt length
37.9 ±17b
crypt
7@7 0.7
LI
20.5
31.3 ±1.2
p<0.01
4.0 ±0.6
12.9
DMH (4)
39.4 ±1.8
8.5 ±0.9
21.8
Control
28.3 ±0.7
3.8 ±0.9
13.7
21.2
15.2
Control (5)@
p<0.018
@
(5)
p<0.0116
p<0.01
DMH (3)
Control (3)
p<0.0120
42.6 ±1 .8
29.0 ±1 .4
p<0.01
9.0 ±0.3
4.4 ±0.8
17.726
DMH (3@
42.1 ±0.5
7.4 ±0.3
DMH (7)
Control (7)
52.7 ±1.5
31.8 ±1.0
7.2 ±0.4
3.5 ±0.2
p < 0.005
p < 0.01
b Mean
C Controls
1682
0.01).
Although
there
was
some
variation
between
the treat
ment periods as to the number of labeled cells within each
of the quarters, analysis of variance revealed that these
differences were not significant (p > 0.05). The analysis of
variance and the Newman-Keuls multiple range test showed
that there were significantly greater numbers of labeled
cells in Quarter I compared to Quarter lb (p < 0.01) and in
Quarter II compared to Quarter III (p < 0.01) for all durations
of treatment.
The effects of treatment on LI in individual segments of
the crypt columns are shown in Chart 3. Covaniance analy
sis of data from control and DMH-treated animals indicated
that the slopes of the 2 lines were the same but that theme
was an overall increase in LI in the expemimentals(p < 0.01).
The distribution of labeled cells from animals treated for 26
weeks was different than that from animals in other treat
ment groups. The results revealed that (a) DMH treatment
increased the number of labeled cells uniformly throughout
the proliferative zone of the crypt; and (b) although the crypt
columns lengthened initially, the relative distribution of the
labeled cells in the cnypts was not altered by DMH treatment
until frank tumor development after 26 weeks. In these later
animals, relatively fewer cells were seen at the base and
relatively more cells were seen at the mouth of the crypt.
Length of the Cell Cycle. The lengths of the cell cycle and
S phase
(DNA
synthesis
time)
were
measured
in the
crypt
cell populations at selected times after DMH treatment
(Chart 4). The values from animals treated with DMH for
various durations were not different; therefore, the values
Labeled cells/
(wk)
(%)2 Treatment
DMH(4)
a Numbers
momsbegan to develop after 20 to 26 weeks. In crypt cob
umns adjacent to developing tumor, the crypt lengths in
creased markedly, whereas comparable increases in the
number of labeled cells did not occur; hence, the LI de
creased in these crypts.
Absolute and Relative Distribution of Labeled Cells in
Crypt Columns. The distribution of labeled cells in each
quarter of the Crypt column is shown in Chart 2. The abso
lute numbers of labeled cells in each quarter of the crypts
were greaten in DMH-tneated animals than in controls (p <
in parentheses,
number
13.7
11.4
this
group
were
mishandled
of animals.
and
are
not
Weeksoft8IG2O26
Treatment
@@I62O@6
First
included.
té162026
Second
Quarters
± SE.
in
j::
2eI62O@6
Third
of the
crypt
Fourth
column
Chart 2. Distribution of the number of cells in quarters of the crypt that
were labeled 1 hr after i.p. injection of [3H]TdR. Each point Is the mean;
bars, SE.; ----,
control animals; —, DMH-treated animals.
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Crypt Cell Dynamics
and Colon Cancer
showing that treatment of mice with DMH for 26 weeks
induces a high incidence of adenocarcinoma in mucosa of
distal colon. Small lesions, consisting of clusters of imnegu
lamlyshaped crypt glands lined with atypical epithelial cells
(focal atypias), appear on the mucosal folds of colon prior
to the development of frank tumor. These lesions commonly
U
occur between the 12th (5, 20) and 15th to 16th (13) weeks
0
of DMH treatment, although a few scattered atypias were
noted after 38 days of treatment
(5, 20). In this study, atypias
were first observed in colonic mucosa after 16 weeks of
DMH treatment. In addition, the present results demon
strate that DMH leads to a marked increase in cellularity in
colonic cnypts several weeks before the appearance
I
segmentsof crypt column
Chart 3. Distribution of the percentage of cells that are labeled 1 hr after
i.p. injection of [3HJTdRin segmentsof crypt columnsfrom control animals
(x- —
- x) and DMH-treated animals after 2, 8, 16, and 20 weeks of treatment
(x—x) and after 26 weeks of treatment ( 0). Each point is the mean; bars,
SE.
long before the appearance
Time (hours after [@@-Thymidineinjection)
Chart4. Lengthof the cell cyclesandthe S phase(DNAsynthesistime)of
the crypt cell populations. Each point is the mean; bars,S.E.; - - - -, control
animals; —, DMH-treated animals.
for all DMH-tneated groups were combined. The S phase
was 7 to 8 hn, and the length of 1 cell cycle was 25 to 26 hr
for crypt cells of colons from both control and DMH-tneated
animals. There was no significant effect of treatment on
these indices.
Rate of Migration In Crypt Columns. Estimates of the
maximum rates of cell migration along the crypt column by
cells in the nonprolifenative compartment of the crypt were
1.1 and 0.7 cell positions/hr for DMH-tneated and control
animals, respectively. This difference reflects the increased
number of mitotic cells per crypt after DMH treatment.
However, because the crypt length also increased after
DMH treatment, the cells had to migrate a greater distance
in the crypt before reaching the mouth of the gland. As a
result the cell transit times through the nonproliferative
zone of the crypt (i.e. , Quarter IV) were comparable in
expenimentals and controls (9 and 10 hn, respectively).
DISCUSSION
The results of the present experiments confirm the work
of Thumnhemret al. (20), Deschner (5), and Maskens (13) in
JUNE 1977
of atyp
ias. The proportion of cells in the elongated crypts that were
actively dividing, i.e. , cells in mitosis or in the S phase of
the cell cycle, also increased, although the relative distnibu
tion of dividing cells within the crypt was not affected.
Further changes in the indices of cell kinetics of mucosal
crypts were not observed until frank tumor had developed
after 26 weeks of DMH treatment. These findings suggest
that the mechanisms that regulate crypt dynamics are “me
set―and that a bangercrypt cell population is established
of adenocarcinoma.
It cannot
be determined from the present results whether these initial
changes are an integral part of the carcinogenic process or
represent a less specific response to DMH.
Chan et al. (2) reported that, following a single in. instilla
tion of DMH, the LI in distal colon increased by 2-fold after
24 hr and by 3-fold after 2 weeks. These changes in the LI
occurred earlier and were greaten in magnitude than those
observed here. Such differences in latency and amplitude of
response of DMH-sensitive cells in colon may be due to the
route of administration and/orthe concentration of DMH.
The eventual development of neoplastic growth in mu
cosa of colon may be dependent in part on disruptions in
the normal distribution
of dividing
cells in the crypt. Epithe
hal cells located in the bower two-thirds of the cobonic crypt
(proliferative zone) undergo DNA synthesis and proliferative
activity (4). These cells migrate toward the cryptal lumen
and cease dividing
as they reach the upper portion
of the
crypt (4, 12). Thumnhemm
et a!. (20) and Lipkin (10) reported
that, in animals treated with DMH for 12 weeks, a greater
number of dividing cells were located in cell positions fur
them from the crypt base than in comparable
controls.
This
alteration was interpreted as evidence for the expansion of
the proliferative zone of the crypt. The results of the present
study support this interpretation in that an absolute en
langement of the proliferative
zone was observed; i.e., more
cell positions in the crypt columns contained mitotically
active cells. However, a relative enlargement of the prolifem
ative zone was not demonstrated because the total number
of cells in the crypt column also increased. Thus, the dis
tance between the highest cells undergoing division and the
mouth of the crypt remained fairly constant. Therefore, the
action of DMH does not alter the relative distribution of
labeled cells in crypts, at least not during the inductive
period prior to the appearance of tumors. This difference in
interpretation between the present study and the work of
Thunnhemret a!. (20) and Lipkin (10) may be a reflection of
the method by which the crypt cells were analyzed. In this
study the LI was calculated from populations of cells in the
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T. C. Richards
quarters and segments of crypt columns, and these popula
tions were normalized to reflect fluctuations
in the total
number of cells in the crypt column. In contrast, Thumnhemn
et at. (20) and Lipkin (10) determined the Lbof individual cell
positions and did not take into account the crypt length.
The present results show that the crypt columns are en
bangedby 2 weeks after initial DMH treatment. Chang and
Nadlem(4) suggested that the balance between cell produc
tion and cell loss is dependent
in part on the maturity of the
experiments. However, it appears that the DMH-induced
transformation to tumor occurs in an altered population of
crypt cells.
ACKNOWLEDGMENTS
I gratefully acknowledge the technical assistance of Patricia Richardson
and the expertadviceof Dr. H. Weitlauf.
cells. An increased crypt length might be expected to result
from
an increase
in cell division
because
less mature
cells
would be located higher in the crypts and would not be lost
until
they
had had time
to mature.
Thus,
lengthening
of the
crypt would continue until sufficient time for maturation
had elapsed, at which point cell bossshould again equal cell
production (14). Under these conditions the population size
would be reset at a higher bevel.
Kanagalingam and Babis (8) postulated that the develop
ment of tumor results from a reduced ability of cells to
repair
DNA damaged
by chronic
exposure
to high doses
of a
carcinogen. Colonic mucosa consists of cells with a short
G phase (10 hr) and a life-span of 100 to 120 hr (3, 4). In
such a tissue errors in DNA could be incorporated into the
next generation of cells if DNA repair is not completed
before cell division occurs. In this way the errors would
accumulate during chronic exposure to the carcinogen.
Enlargement
of the population
may play a significant
mole in
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that
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formation
is increased
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is effected
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‘promoters'â€ãcting on the altered cells. That a
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treatment. Thus, the action of DMH may require 2 steps: (a)
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step that stimulates
alterations
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Fig. 1. Section of mucosa of colon from control animal. The nuclei of the crypt columns are more basophilic than those in Fig. 2. H & E, x 60.
Fig. 2. Sectionof mucosaof colon from animaltreatedwith DMHfor 2 weeks.Notethe elongatedcrypt columnsand increasedcellularityof the lamina
propriacompared tothatinFig.1.H & E, x60.
Fig. 3. Section of mucosa of colon from animal treated with DMH for 16 weeks. Note the focal atypia at the tip of the mucosal fold. H & E, x 60.
Fig. 4. Section of mucosa and tumor of colon from animal treated with DMH for 26 weeks. This particular intramucosal carcinoma had not invaded the
submucosa. Goblet cells, well demonstrated in the adjacent mucosa, are markedly reduced in the tumor. Periodic acid-Schiff, x 60.
1684
CANCER
RESEARCH
VOL. 37
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1685
Early Changes in the Dynamics of Crypt Cell Populations in
Mouse Colon following Administration of 1,2-Dimethylhydrazine
Thomas C. Richards
Cancer Res 1977;37:1680-1685.
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